Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby

Abstract

The present invention relates generally to the field of investigational bioinformatics and more particularly to secondary structure defining databases. The present invention further relates to methods for interrogating a database as a source of molecular masses of known bioagents for comparing against the molecular mass of an unknown or selected bioagent to determine either the identity of the selected bioagent, and/or to determine the origin of the selected bioagent. The identification of the bioagent is important for determining a proper course of treatment and/or irradication of the bioagent in such cases as biological warfare. Furthermore, the determination of the geographic origin of a selected bioagent will facilitate the identification of potential criminal identity.

1. A method for providing bioagent characterizing information comprising:
 a) measuring or calculating with a mass spectrometer a plurality of molecular masses corresponding to a plurality of amplification products wherein the amplification products are obtained by amplification of at least one target sequence region of a bioagent nucleic acid gene sequence using a primer pair that hybridizes to the at least one target sequence region of at least nineteen bioagents; said target sequence region comprising two conserved regions that are hybridizable with the primer pair and that flank a variable region that uniquely varies between at least eight bioagents; 
 b) interrogating a database stored on a computer readable medium with an identification query, wherein said identification query comprises comparison of the measured molecular mass data of step a) with said database; said database comprising molecular masses calculated or measured for the target sequence regions for at least nineteen bioagents and each of the calculated or measured molecular masses is indexed to bioagent characterizing information; 
 c) delivering from said database a response comprising bioagent characterization information generated by the comparison of said measured molecular masses of step a) and said calculated or measured molecular masses of step b) thereby identifying a bioagent associated with an amplification product of step a). 
 
     
     
       2. The method of  claim 1  wherein the nucleic acid gene sequence encodes ribosomal RNA or a protein involved in translation, replication, recombination, repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, or secretion. 
     
     
       3. The method of  claim 1  wherein the bioagent characterizing information is a genus name. 
     
     
       4. The method of  claim 3  wherein the genus name is  Acinetobacter, Aeromonas, Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio , or  Yersinia.    
     
     
       5. The method of  claim 1  wherein the bioagent characterizing information is a species name. 
     
     
       6. The method of  claim 1  wherein the bioagent characterizing information is a strain name. 
     
     
       7. The method of  claim 1  wherein the response is delivered via a network. 
     
     
       8. The method of  claim 7  wherein the network is a local area network, a wide area network, or the internet. 
     
     
       9. The method of  claim 1  wherein said mass spectrometer is an electrospray Fourier transform ion cyclotron resonance mass spectrometer or an electrospray time-of-flight mass spectrometer. 
     
     
       10. The method of  claim 1  wherein the primer pair comprises at least one modified nucleobase. 
     
     
       11. The method of  claim 10  wherein the modified nucleobase comprises 2,6-diaminopurine, propyne C, propyne T, phenoxazine, or G-clamp. 
     
     
       12. The method of  claim 1  wherein said bioagent is a biological warfare agent. 
     
     
       13. The method of  claim 12  wherein the biological warfare agent comprises  Bacillus anthracis, Yersinia pestis, Franciscella tularensis, Brucella suis, Brucella abortus, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomalleii, Salmonella typhi, Rickettsia typhii, Rickettsia prowasekii, Coxiella burnetii, Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila , or  Vibrio cholerae.    
     
     
       14. The method of  claim 1  wherein said bioagent is a bacterium, virus, fungus or protozoan. 
     
     
       15. The method of  claim 14  wherein the bioagent is arenavirus, bunyavirus, mononegavirales, picornavirus, astrovirus, calcivirus, nidovirales, flavivirus or togavirus. 
     
     
       16. A method for providing bioagent characterizing information comprising:
 a) measuring or calculating with a mass spectrometer a plurality of molecular masses corresponding to a plurality of amplification products wherein the amplification products are obtained by amplification of at least one target sequence of a bioagent nucleic acid gene sequence using a primer pair said target sequence region comprising two conserved regions that are hybridizable with the primer pair and that flank a variable region that uniquely varies between bioagents; 
 b) calculating base compositions from said molecular mass measurements wherein said base compositions identify the number of A residues, C residues, T residues, G residues, U residues, analogues thereof and mass tag residues thereof; 
 c) interrogating a database stored on a computer readable medium with an identification query, wherein said identification query comprises comparison of the base composition data from step b) with said database; said database comprising base composition data calculated or measured for the target sequence regions for at least nineteen bioagents and each of the calculated or measured base compositions is indexed to bioagent characterizing information; and 
 d) delivering from said database a response comprising bioagent characterization information generated by the comparison of said measured base compositions of step b) and the calculated or measured base compositions of step c) thereby identifying a bioagent associated with an amplification product of step a). 
 
     
     
       17. The method of  claim 16  wherein the nucleic acid gene sequence encodes ribosomal RNA or a protein involved in translation, replication, recombination, repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, or secretion. 
     
     
       18. The method of  claim 16  wherein the bioagent characterizing information is a genus name. 
     
     
       19. The method of  claim 18  wherein the genus name is  Acinetobacter, Aeromonas, Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio , or  Yersinia.    
     
     
       20. The method of  claim 16  wherein the bioagent characterizing information is a species name. 
     
     
       21. The method of  claim 16  wherein the bioagent characterizing information is a strain name. 
     
     
       22. The method of  claim 16  wherein the response is delivered via a network. 
     
     
       23. The method of  claim 22  wherein the network is a local area network, a wide area network, or the internet. 
     
     
       24. The method of  claim 16  wherein said mass spectrometer is an electrospray Fourier transform ion cyclotron resonance mass spectrometer or an electrospray time-of-flight mass spectrometer. 
     
     
       25. The method of  claim 16  wherein the primer pair comprises at least one modified nucleobase. 
     
     
       26. The method of  claim 25  wherein the modified nucleobase comprises 2,6-diaminopurine, propyne C, propyne T, phenoxazine, or G-clamp. 
     
     
       27. The method of  claim 16  wherein said bioagent is a biological warfare agent. 
     
     
       28. The method of  claim 27  wherein the biological warfare agent comprises  Bacillus anthracis, Yersinia pestis, Franciscella tularensis, Brucella suis, Brucella abortus, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomalleii, Salmonella typhi, Rickettsia typhii, Rickettsia prowasekii, Coxiella burnetii, Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila , or  Vibrio cholerae.    
     
     
       29. The method of  claim 16  wherein said bioagent is a bacterium, virus, fungus or protozoan. 
     
     
       30. The method of  claim 29  wherein the bioagent is arenavirus, bunyavirus, mononegavirales, picornavirus, astrovirus, calcivirus, nidovirales, flavivirus or togavirus.